A number of medical diagnostic procedures, including PET and SPECT utilize radiolabeled compounds. PET and SPECT are very sensitive techniques and require small quantities of radiolabeled compounds, called tracers. The labeled compounds are transported, accumulated and converted in vivo in exactly the same way as the corresponding non-radioactively compound. Tracers, or probes, can be radiolabeled with a radionuclide useful for PET imaging, such as 11C, 13N, 15O, 18F, 61Cu, 62Cu, 64Cu, 67Cu, 68Ga, 124I, 125I and 131I, or with a radionuclide useful for SPECT imaging, such as 99Tc, 75Br, 61Cu, 153Gd, 125I, 131I and 32P.
PET creates images based on the distribution of molecular imaging tracers carrying positron-emitting isotopes in the tissue of the patient. The PET method has the potential to detect malfunction on a cellular level in the investigated tissues or organs. PET has been used in clinical oncology, such as for the imaging of tumors and metastases, and has been used for diagnosis of certain brain diseases, as well as mapping brain and heart function. Similarly, SPECT can be used to complement any gamma imaging study, where a true 3D representation can be helpful, for example, imaging tumor, infection (leukocyte), thyroid, or bones.
Angiogenesis plays a vital role in tumor growth and metastatic spread. Tumor angiogenesis is a multi-step process characterized by chemotactic and mitogenic response of endothelial cells to angiogenic growth factors, proteolytic degradation of extracellular matrix, and modulation of endothelial cell interaction with extracellular matrix mediated by integrin receptors. Each of these steps may represent a potential target for the development of tumor angiogenic and metastatic diagnostics.
Integrins are a family of membrane-spanning adhesion receptors composed of noncovalently linked α and β subunits, which combine to form a variety of heterodimers with different ligand recognition properties. Several integrins have been shown to interact with polypeptide domains containing the Arg-Gly-Asp (“RGD”) amino acid sequence present in various extracellular matrix-associated adhesive glycoproteins. Besides cell adhesion to extracellular matrix, integrins also mediate intracellular events that control cell migration, proliferation, and survival.
One member of the integrin family, αvβ3 integrin, plays a key role in angiogenesis. It interacts with several extracellular matrix proteins, such as vitronectin, fibrinogen, fibronectin, thrombin, and thrombospondin, and cooperates with molecules such as metalloproteases, growth factors, and their receptors. Due to its numerous functions and relatively limited cellular distribution, αvβ3 integrin represents an attractive target for diagnostic and therapeutic intervention. In addition, findings that several extracellular matrix proteins, such as vitronectin, fibrinogen, and thrombospondin interact with integrins via the RGD sequence has lead to the development of synthetic linear and cyclic peptides containing RGD sequence for integrin targeting. See e.g. DE 197 25 368, U.S. Pat. No. 5,849,692, U.S. Pat. No. 6,169,072, U.S. Pat. No. 6,566,491, U.S. Pat. No. 6,610,826, and WO 2005/111064.
It has also been demonstrated in a number of murine tumor models that radiolabeled peptides containing the RGD motif can be used for non-invasive investigation of αVβ3 integrin expression. The development of noninvasive methods to visualize and quantify integrin αvβ3 expression in vivo appears to be closely related to the success of antiangiogenic therapy based on integrin antagonism. Precise documentation of integrin receptor levels allows appropriate selection of patients who will most likely benefit from an anti-integrin treatment regimen. Imaging can also be used to provide an optimal dosage and time course for treatment based on receptor occupancy studies. In addition, imaging integrin expression is used to evaluate anti-integrin treatment efficacy and to develop new therapeutic drugs with favorable tumor targeting and in vivo kinetics.
Kessler and co-workers [1] developed the pentapeptide cyclo(-Arg-Gly-Asp-D-Phe-Val-) (“c(RGDfV)”) which showed high affinity and selectivity for integrin αvβ3. To date, most integrin αvβ3 targeted PET studies have been focused on radiolabeling of c(RGDfV)-based antagonists due to its high binding affinity (nanomolar to subnanomolar range for monomeric and multimeric c(RGDfV) respectively). In the late 1990's, the monomeric peptide c(RGDyV) was labeled by Haubner et al. [2] with 125I. This tracer revealed receptor-specific tumor uptake in vivo. However, the labeled peptide had rapid tumor washout and unfavorable hepatobiliary excretion resulting from its high lipophilicity, which limited its further application. Glycosylation on the lysine side chain of a similar RGD peptide, c(RGDyK), decreased lipophilicity and hepatic uptake [3]. A glycopeptide based on c(RGDfK), [18F]galacto-RGD, was then synthesized:

It was demonstrated that [18F]galacto-RGD exhibited integrin αvβ3-specific tumor uptake in integrin-positive M21 melanoma xenograft model [4-6, see also 19]. Moreover, [18F]galacto-RGD was sensitive enough for visualization of integrin αvβ3 expression resulting exclusively from the tumor vasculature using an A431 human squamous cell carcinoma model, in which the tumor cells are integrin negative. Initial clinical trials in healthy volunteers and a limited number of cancer patients revealed that this tracer could be safely administered to patients and was able to delineate certain lesions that were integrin-positive with reasonable contrast.
[18F]Galacto-RGD currently represents one promising integrin marker for PET imaging of angiogenesis. As a monomeric RGD peptide tracer, it has relatively low tumor targeting efficacy; clinical use of this tracer is severely limited because of its relatively low integrin binding affinity, modest tumor standard uptake values, and unfavorable pharmacokinetic behavior. Therefore, tumors with low integrin expression level may not be detectable. In addition, prominent activity accumulation in the liver, kidneys, spleen, and intestines was observed in both preclinical models and human studies. As a result, it was difficult to visualize lesions in the abdomen. This tracer is also very difficult to synthesize, thereby limiting its availability.
Conjugation of PEG (poly(ethyleneglycol)) (“PEGylation”) has been shown to improve many properties of peptides and proteins, including plasma stability, immunogenicity, and pharmacokinetics. Chen et al. [7-9] conjugated RGD-containing peptides with PEG moieties of different sizes and synthesized radioiodinated, 18F- and 64Cu-labeled derivatives. PEGylation demonstrated an effect on the pharmacokinetics, tumor uptake and retention of the RGD peptides, which seem to depend strongly on the nature of lead structure and on the size of the PEG moiety. Additional strategies for improving pharmacokinetic behavior as well as tumor uptake and retention pattern of peptides with an RGD motif include introduction of hydrophilic amino acids and multimerisation of RGD.